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Mucormycosis is a life-threatening fungal infection caused by fungi of the order Mucorales. It usually affects people with weakened immune systems, such as those with uncontrolled diabetes, acquired immunodeficiency syndrome, iatrogenic immunosuppression, and hematological malignancies, as well as individuals who have had organ transplants. The type of mucormycosis a person suffers from is often determined by their underlying conditions. The most common types are rhino-cerebral mucormycosis, pulmonary mucormycosis, cutaneous mucormycosis, cerebral mucormycosis, gastrointestinal mucormycosis, and disseminated mucormycosis. The incidence of mucormycosis has been increasing over the years, with an overall mortality rate of 54%. Recent cases have shown a correlation between COVID-19 and mucormycosis. Using anti-inflammatory drugs to combat the cytokine storm associated with COVID-19 can weaken the immune system, making individuals more susceptible to opportunistic fungal infections like mucormycosis. Underlying health conditions further exacerbate the condition. This study reviewing 198 cases of mucormycosis and conducting a meta-analysis found that post-COVID-19 patients most commonly developed rhino-orbital-cerebral mucormycosis, followed by pulmonary and gastrointestinal mucormycosis. The study also identified diabetes as the most common underlying factor contributing to the development of mucormycosis in post-COVID-19 patients, followed by hypertension and obesity. The study also examined the influence of age, affected organs, and the use of certain drugs on the development of mucormycosis. Age was found to be a significant factor in the infection. This meta-analysis is one of the first to compare post-COVID mucormycosis cases with those from the pre-COVID era. The hope is that this study and analysis will help identify the determinants of mucormycosis in post-COVID patients and aid the scientific community in finding a solution to this problem. [ABSTRACT FROM AUTHOR]

Loop-mediated isothermal amplification (LAMP) is a rapid, state-of-the-art DNA amplification technology, used primarily for the quick diagnosis and early identification of microbial infection, caused by pathogens such as virus, bacteria and malaria. A target DNA can be amplified within 30 min using the LAMP reaction, taking place at a steady temperature. The LAMP method uses four or six primers to bind eight regions of a target DNA and has a very high specificity. The devices used for conducting LAMP are usually simple since the LAMP method is an isothermal process. When LAMP is coupled with Reverse Transcription (RT), it allows direct detection of RNA in a sample. This greatly enhances the efficiency of diagnosis of RNA viruses in a sample. Recently, the rampant spread of COVID-19 demanded such a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. Loop-mediated isothermal amplification (LAMP) assays are not only used for the detection of microbial pathogens, but there are various other applications such as detection of genetic mutations in food and various organisms. In this review, various implementations of RT-LAMP techniques would be discussed. [ABSTRACT FROM AUTHOR]

In Mycobacterium tuberculosis, hspR is the last gene of the dnaKJE operon. It encodes the repressor HspR, which regulates the expression from this operon by binding to a consensus upstream sequence known as HAIR (HspR-associated inverted repeats). Previous investigations in the related Gram-positive bacterium Streptomyces coelicolor have revealed that DnaK acts as a co-repressor for HspR. In this investigation, a similar situation was encountered using the corresponding mycobacterial pair. However, the novel feature unearthed in this study is that the mycobacterial GroELs, GroEL1 and GroEL2, considerably stimulate the HAIR-binding activity of the HspR-DnaK combination. That these GroELs play a role in the folding process was evident from the observation that when heat- or chemically denatured HspR was renatured, the protein gained optimal activity only if one of these GroEL class chaperones was present along with DnaK. The renaturation process was found to be dependent on ATP hydrolysis. The DnaK-dependent DNA-binding activity of HspR could also be stimulated by DnaJ, but GrpE, which is known to release DnaK-bound substrates, was found to be inhibitory. The results of this study suggest that protein folding plays a substantial role in the activation of HspR following heat shock and that DnaK may be involved in two ways--first, as a chaperone acting in concert with GroEL and/or DnaJ and second, as a co-represser bound to HspR.

Circular permutation is a powerful tool to test the role of topology in protein folding and function. Previous methods for generating circular permutants were based on rearranging gene elements using restriction enzymes-based cloning. Here, we present a Restriction Free (RF) approach to achieve circular permutation which is faster and more cost-effective. [ABSTRACT FROM AUTHOR]

HspR is a repressor known to control expression of heat shock operons in a number of Eubacteria. In mycobacteria and in several other actinobacteria, this protein is synthesized from the dnaKJE-hspR operon. Previous investigations revealed that HspR binds to the operon promoter, thereby controlling its expression in an autoregulatory manner. DnaK, which is a product of the same operon, further aids this autoregulatory process by stimulating the operator binding activity of HspR. The molecular mechanism by which DnaK assists HspR in executing its function is not clearly understood. In this study, it has been shown that DnaK can augment DNA binding activity of HspR by two mechanisms: (i) DnaK can restore the activity of completely denatured HspR by forming a complex with it, and (ii) DnaK can prevent thermal instability of HspR renatured by other means. Unlike the first mechanism, the latter function does not involve complex formation. The C-terminal hydrophobic tail of HspR was found to play a significant role in determining its thermal stability and DnaK dependence properties. A deletion mutant in which this region is removed does not respond to thermal stress and functions independent of DnaK. The hydrophobic C-terminal tails of HspRs of Mycobacterium tuberculosis and related Actinomycetales therefore may have evolved to make these HspRs more sensitive to thermal stress and, at the same time, subject to regulation by DnaK. [ABSTRACT FROM AUTHOR]

The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)